TY - JOUR A1 - Kindzierski, V. A1 - Raschke, Silvia A1 - Knabe, Nicole A1 - Siedler, F. A1 - Scheffer, B. A1 - Pflüger-Grau, K. A1 - Pfeiffer, F. A1 - Oesterhelt, D. A1 - Marin-Sanguino, A. A1 - Kunte, Hans-Jörg T1 - Osmoregulation in the halophilic bacterium halomonas elongata: A case study for integrative systems biology N2 - Halophilic bacteria use a variety of osmoregulatory methods, such as the accumulation of one or more compatible solutes. The wide diversity of compounds that can act as compatible solute complicates the task of understanding the different strategies that halophilic bacteria use to cope with salt. This is specially challenging when attempting to go beyond the pathway that produces a certain compatible solute towards an understanding of how the metabolic network as a whole addresses the problem. Metabolic reconstruction based on genomic data together with Flux Balance Analysis (FBA) is a promising tool to gain insight into this problem. However, as more of these reconstructions become available, it becomes clear that processes predicted by genome annotation may not reflect the processes that are active in vivo. As a case in point, E. coli is unable to grow aerobically on citrate in spite of having all the necessary genes to do it. It has also been shown that the realization of this genetic potential into an actual capability to metabolize citrate is an extremely unlikely event under normal evolutionary conditions. Moreover, many marine bacteria seem to have the same pathways to metabolize glucose but each species uses a different one. In this work, a metabolic network inferred from genomic annotation of the halophilic bacterium Halomonas elongata and proteomic profiling experiments are used as a starting point to motivate targeted experiments in order to find out some of the defining features of the osmoregulatory strategies of this bacterium. This new information is then used to refine the network in order to describe the actual capabilities of H. elongata, rather than its genetic potential. KW - Halomonas elongata KW - Systems biology KW - Flux balance analysis KW - Proteomic analysis PY - 2017 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-391172 DO - https://doi.org/10.1371/journal.pone.0168818 SN - 1932-6203 VL - 12 IS - 1 SP - Article e0168818, 1 EP - 22 AN - OPUS4-39117 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Moghaddam, J. A. A1 - Böhringer, N. A1 - Burdziak, A. A1 - Kunte, Hans-Jörg A1 - Galinski, E. A. A1 - Schäberle, T. F. T1 - Different strategies of osmoadaptation in the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 N2 - Only a few myxobacteria are known to date that are classified as marine, owing to their salt dependency. In this study, the salt tolerance mechanism of these bacteria was investigated. To this end, a growth medium was designed in which the mutated Escherichia coli strain BKA13 served as sole food source for the predatory, heterotrophic myxobacteria. This enabled measurement of the osmolytes without any background and revealed that the closely related strains Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 developed different strategies to handle salt stress. Ple. pacifica SIR-1, which was grown between 1 and 4% NaCl, relies solely on the accumulation of amino acids, while Enh. salina SWB007, which was grown between 0.5 and 3% NaCl, employs, besides betaine, hydroxyectoine as the major compatible solute. In accordance with this analysis, only in the latter strain was a locus identified that codes for genes corresponding to the biosynthesis of betaine, ectoine and hydroxyectoine. KW - Osmoadaptation PY - 2016 DO - https://doi.org/10.1099/mic.0.000250 VL - 162 SP - 651 EP - 661 PB - SOC General Microbiology CY - Berks, UK AN - OPUS4-35782 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Meyer, Susann A1 - Schröter, Maria-Astrid A1 - Hahn, Marc Benjamin A1 - Solumon, Tihomir A1 - Strum, Heinz A1 - Kunte, Hans-Jörg T1 - Ectoine can enhance structural changes in DNA in vitro N2 - Strand breaks and conformational changes of DNA have consequences for the physiological role of DNA. The natural protecting molecule ectoine is beneficial to entire bacterial cells and biomolecules such as proteins by mitigating detrimental effects of environmental stresses. It was postulated that ectoine-like molecules bind to negatively charged spheres that mimic DNA surfaces. We investigated the effect of ectoine on DNA and whether ectoine is able to protect DNA from damages caused by ultraviolet radiation (UV-A). In order to determine different isoforms of DNA, agarose gel electrophoresis and atomic force microscopy experiments were carried out with plasmid pUC19 DNA. Our quantitative results revealed that a prolonged incubation of DNA with ectoine leads to an increase in transitions from supercoiled (undamaged) to open circular (single-strand break) conformation at pH 6.6. The effect is pH dependent and no significant changes were observed at physiological pH of 7.5. After UV-A irradiation in ectoine solution, changes in DNA conformation were even more pronounced and this effect was pH dependent. We hypothesize that ectoine is attracted to the negatively charge surface of DNA at lower pH and therefore fails to act as a stabilizing agent for DNA in our in vitro experiments. KW - Ectoine KW - DNA KW - UV radiation PY - 2017 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-413139 DO - https://doi.org/10.1038/s41598-017-07441-z VL - 7 IS - 1 SP - Article 7170, 1 EP - 10 AN - OPUS4-41313 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kuhlmann, S. A1 - Terwisscha van Scheltinga, A. C. A1 - Bienert, Ralf A1 - Kunte, Hans-Jörg A1 - Ziegler, C. M. T1 - 1.55 Å Structure of the Ectoine Binding Protein TeaA of the Osmoregulated TRAP-Transporter TeaABC from Halomonas elongata N2 - TeaABC from the moderate halophilic bacterium Halomonas elongata belongs to the tripartite ATP-independent periplasmic transporters (TRAP-T), a family of secondary transporters functioning in conjunction with periplasmic substrate binding proteins. TeaABC facilitates the uptake of the compatible solutes ectoine and hydroxyectoine that are accumulated in the cytoplasm under hyperosmotic stress to protect the cell from dehydration. TeaABC is the only known TRAP-T activated by osmotic stress. Currently, our knowledge on the osmoregulated compatible solute transporter is limited to ABC transporters or conventional secondary transporters. Therefore, this study presents the first detailed analysis of the molecular mechanisms underlying substrate recognition of the substrate binding protein of an osmoregulated TRAP-T. In the present study we were able to demonstrate by isothermal titration calorimetry measurements that TeaA is a high-affinity ectoine binding protein (Kd = 0.19 μM) that also has a significant but somewhat lower affinity to hydroxyectoine (Kd = 3.8 μM). Furthermore, we present the structure of TeaA in complex with ectoine at a resolution of 1.55 Å and hydroxyectoine at a resolution of 1.80 Å. Analysis of the TeaA binding pocket and comparison of its structure to other compatible solute binding proteins from ABC transporters reveal common principles in compatible solute binding but also significant differences like the solvent-mediated specific binding of ectoine to TeaA. KW - Halophilic Bacteria KW - Halomonas elongata KW - Substrate binding protein KW - TRAP-transporter KW - TeaABC KW - osmoregulation KW - compatible solutes KW - ectoine KW - hydroxyectoine PY - 2008 DO - https://doi.org/10.1021/bi8006719 SN - 0006-2960 SN - 1520-4995 VL - 47 IS - 36 SP - 9475 EP - 9485 PB - ACS Publ. CY - Washington, DC AN - OPUS4-18560 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Osterloh, Kurt A1 - Wrobel, Norma A1 - Kunte, Hans-Jörg A1 - Zscherpel, Uwe A1 - Ewert, Uwe T1 - Making the world a safer place - some know-how already exists T2 - MATEST 2009 CY - Cavtat/Dubrovnik, Croatia DA - 2009-09-23 KW - Existing hazards KW - Threat detection KW - Interdisciplinary approach KW - Existing safety rules and procedures KW - Bridging safety and security KW - Common definitions PY - 2009 SN - 978-953-7283-03-2 IS - #10 SP - 1 EP - 7 AN - OPUS4-20710 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schweikhard, E.S. A1 - Kuhlmann, S.I. A1 - Kunte, Hans-Jörg A1 - Grammann, K. A1 - Ziegler, C. M. T1 - Structure and function of the universal stress protein TeaD and its role in regulating the ectoine transporter TeaABC of halomonas elongata DSM 2581T N2 - The halophilic bacterium Halomonas elongata takes up the compatible solute ectoine via the osmoregulated TRAP transporter TeaABC. A fourth orf (teaD) is located adjacent to the teaABC locus that encodes a putative universal stress protein (USP). By RT-PCR experiments we proved a cotranscription of teaD along with teaABC. Deletion of teaD resulted in an enhanced uptake for ectoine by the transporter TeaABC and hence a negative activity regulation of TeaABC by TeaD. A transcriptional regulation via DNA binding could be excluded. ATP binding to native TeaD was shown by HPLC, and the crystal structure of TeaD was solved in complex with ATP to a resolution of 1.9 Å by molecular replacement. TeaD forms a dimer–dimer complex with one ATP molecule bound to each monomer, which has a Rossmann-like α/β overall fold. Our results reveal an ATP-dependent oligomerization of TeaD, which might have a functional role in the regulatory mechanism of TeaD. USP-encoding orfs, which are located adjacent to genes encoding for TeaABC homologues, could be identified in several other organisms, and their physiological role in balancing the internal cellular ectoine pool is discussed. PY - 2010 DO - https://doi.org/10.1021/bi9017522 SN - 0006-2960 SN - 1520-4995 VL - 49 IS - 10 SP - 2194 EP - 2204 PB - ACS Publ. CY - Washington, DC AN - OPUS4-21915 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kraegeloh, A. A1 - Amendt, B. A1 - Kunte, Hans-Jörg T1 - Potassium Transport in a Halophilic Member of the Bacteria Domain: Identification and Characterization of the K+ Uptake Systems TrkH and TrkI from Halomonas elongata DSM 2581T N2 - The halophilic bacterium Halomonas elongata accumulates K+, glutamate, and the compatible solute ectoine as osmoprotectants. By functional complementation of Escherichia coli mutants defective in K+ uptake, we cloned three genes that are required for K+ uptake in H. elongata. Two adjacent genes, named trkA (1,374 bp) and trkH (1,449 bp), were identified on an 8.5-kb DNA fragment, while a third gene, called trkI (1,479 bp), located at a different site in the H. elongata chromosome, was found on a second 8.5-kb fragment. The potential protein expressed by trkA is similar to the cytoplasmic NAD+/NADH binding protein TrkA from E. coli, which is required for the activity of the Trk K+ uptake system. The deduced amino acid sequences of trkH and trkI showed significant identity to the transmembrane protein of Trk transporters. K+ transport experiments with trkH and trkI mutants of H. elongata revealed that TrkI exhibits a Km value of 1.12 mM, while the TrkH system has a half-saturation constant of 3.36 mM. Strain KB12, relying on TrkH alone, accumulated K+ with a lower Vmax and required a higher K+ concentration for growth in highly saline medium than the wild type. Strain KB15, expressing only TrkI, showed the same phenotype and the same K+ transport kinetics as the wild type, proving that TrkI is the main K+ transport system in H. elongata. In the absence of both transporters TrkH and TrkI, K+ accumulation was not detectable. K+ transport was also abolished in a trkA deletion mutant, indicating that TrkI and TrkH depend on one type of TrkA protein. Reverse transcriptase PCR experiments and Northern hybridization analyses of the trkAH locus revealed cotranscription of trkAH as well as a monocistronic transcript with only trkA. KW - Potassium transport KW - Osmoregulation KW - Halophilic Bacteria KW - Halomonas elongata KW - TrkH KW - TrkA PY - 2005 DO - https://doi.org/10.1128/JB.187.3.1036-1043.2005 SN - 0021-9193 SN - 1098-5530 VL - 187 IS - 3 SP - 1036 EP - 1043 PB - American Society for Microbiology CY - Washington, DC AN - OPUS4-6294 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Kunte, Hans-Jörg ED - Gunde-Cimerman, N. ED - Oren, A. ED - Plemenitas, A. T1 - K+ Transport and its Role for Osmoregulation in a Halophilic Member of the Bacteria Domain: Characterization of the K+ Uptake Systems from Halomonas elongata KW - Osmoregulation KW - Potassium KW - Glutamate KW - Potassium transport KW - TrkH KW - TrkA KW - Trkl KW - Halomonas elongata KW - Halophilic Bacteria PY - 2005 SN - 1-402-03632-9 N1 - Serientitel: Cellular Origin, Life in Extreme Habitats and Astrobiology – Series title: Cellular Origin, Life in Extreme Habitats and Astrobiology IS - 9 SP - 289 EP - 300 PB - Springer CY - Berlin AN - OPUS4-11668 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kunte, Hans-Jörg T1 - Osmoregulation in Bacteria: Compatible Solute Accumulation and Osmosensing N2 - Bacteria and Archaea have developed two basic mechanisms to cope with osmotic stress, the salt-in-cytoplasm mechanism, and the organic-osmolyte mechanism. Organic osmolytes or so-called compatible solutes can be accumulated in molar concentration in the cytoplasm and allow for the adaptation of bacterial cells to varying salt concentrations. The biosynthetic pathways of compatible solutes and different compatible solute transport systems are described. A model for osmoregulatory compatible solute accumulation is introduced. KW - Archaea KW - Compatible solutes KW - Halophilic KW - Halotolerant KW - Osmoregulated transporter PY - 2006 DO - https://doi.org/10.1071/EN06016 SN - 1448-2517 SN - 1449-8979 VL - 3 IS - 2 SP - 94 EP - 99 PB - CSIRO Publishing CY - Collingwood, Vic. AN - OPUS4-14258 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kunte, Hans-Jörg T1 - Osmoregulation in halophilic bacteria: Synthesis and transport of osmoprotectants in halomonas elongata T2 - Max-Planck-Institut für Biochemie CY - Planegg, Germany DA - 2006-04-26 PY - 2006 AN - OPUS4-14156 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kunte, Hans-Jörg T1 - Mechanismen der Anpassung an Wasserstress: Synthese und Tansport von Zellschutzstoffen in halophilen Bacteria T2 - Humboldt Universität CY - Berlin, Germany DA - 2006-12-19 PY - 2006 AN - OPUS4-14157 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Marinelli, F. A1 - Kuhlmann, S.I. A1 - Grell, E. A1 - Kunte, Hans-Jörg A1 - Ziegler, C. M. A1 - Faraldo-Gómez, J.D. T1 - Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins N2 - Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na+-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581T, and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former. KW - Binding thermodynamics KW - Periplasmic binding protein KW - Secondary transporter KW - ABC transporter KW - Replica-exchange metadynamics PY - 2011 DO - https://doi.org/10.1073/pnas.1112534108 SN - 0027-8424 SN - 1091-6490 VL - 108 IS - 49 SP - E1285 EP - E1292 PB - National Academy of Sciences CY - Washington, DC AN - OPUS4-25049 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -